Neuroblastoma is an important pediatric cancer as it contributes disproportionately to childhood disease-related morbidity and mortality. We have recently discovered that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. This project will extend our work focused on the overriding hypothesis that ALK is a critical neuroblastoma oncogene and that activation of this cell surface kinase is a tractable therapeutic target. We propose three Specific Aims to validate this hypothesis and extend this work towards new therapeutic strategies in the clinic. First, we will characterize the full spectrum and frequency of germline and somatic DNA alterations (mutation, amplification, translocation) leading to ALK activation using a fully annotated set of 1500 sporadic neuroblastoma tumors obtained at diagnosis, all with available matched germline DNA, and a large set of human neuroblastoma derived cell lines. Second, we will identify the functionally relevant ALK mutations that contribute to the neuroblastoma oncogenic phenotype and examine how these mutations differentially activate downstream signaling pathways. We will determine the malignant transforming properties of all mutations identified in Aim 1 by forcibly over expressing ALK mutants to neural crest-derived retinal pigment epithelial cells (RPE1). To understand the mechanism for malignant transformation, we will survey the downstream signaling pathways activated in ALK mutant and wild-type cells. Finally, we will determine the varying sensitivity of different ALK mutations to pharmacologic inhibition, work that should provide the impetus for developing therapeutic strategies aimed at inhibiting ALK-mediated signaling in the clinic. Preclinical evaluations of anti-tumor efficacy will be designed to quickly develop the rationale necessary to move discoveries in this project to early Phase clinical trials in children with neuroblastoma.